Characterization of Ethiopian Wheat Germplasm for Resistance to Four Puccinia graminis f. sp. tritici Races Facilitated by Single-Race Nurseries.

In Ethiopia, breeding rust resistant wheat cultivars is a priority for wheat production. A stem rust epidemic during 2013 to 2014 on previously resistant cultivar Digalu highlighted the need to determine the effectiveness of wheat lines to multiple races of Puccinia graminis f. sp. tritici in Ethiopia. During 2014 and 2015, we evaluated a total of 97 bread wheat and 14 durum wheat genotypes against four P. graminis f. sp. tritici races at the seedling stage and in single-race field nurseries. Resistance genes were postulated using molecular marker assays. Bread wheat lines were resistant to race JRCQC, the race most virulent to durum wheat. Lines with stem rust resistance gene Sr24 possessed the most effective resistance to the four races. Only three lines with adult plant resistance possessed resistance effective to the four races comparable with cultivars with Sr24. Although responses of the wheat lines across races were positively correlated, wheat lines were identified that possessed adult plant resistance to race TTKSK but were relatively susceptible to race TKTTF. This study demonstrated the importance of testing wheat lines for response to multiple races of the stem rust pathogen to determine if lines possessed non-race-specific resistance. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genotype-by-sequencing facilitates genetic mapping of a stem rust resistance locus in Aegilops umbellulata, a wild relative of cultivated wheat.

BACKGROUND: Wild relatives of wheat play a significant role in wheat improvement as a source of genetic diversity. Stem rust disease of wheat causes significant yield losses at the global level and stem rust pathogen race TTKSK (Ug99) is virulent to most previously deployed resistance genes. Therefore, the objective of this study was to identify loci conferring resistance to stem rust pathogen races including Ug99 in an Aegilops umbelluata bi-parental mapping population using genotype-by-sequencing (GBS) SNP markers. RESULTS: A bi-parental F2:3 population derived from a cross made between stem rust resistant accession PI 298905 and stem rust susceptible accession PI 542369 was used for this study. F2 individuals were evaluated with stem rust race TTTTF followed by testing F2:3 families with races TTTTF and TTKSK. The segregation pattern of resistance to both stem rust races suggested the presence of one resistance gene. A genetic linkage map, comprised 1,933 SNP markers, was created for all seven chromosomes of Ae. umbellulata using GBS. A major stem rust resistance QTL that explained 80% and 52% of the phenotypic variations for TTTTF and TTKSK, respectively, was detected on chromosome 2U of Ae. umbellulata. CONCLUSION: The novel resistance gene for stem rust identified in this study can be transferred to commercial wheat varieties assisted by the tightly linked markers identified here. These markers identified through our mapping approach can be a useful strategy to identify and track the resistance gene in marker-assisted breeding in wheat.

Precisely mapping a major gene conferring resistance to Hessian fly in bread wheat using genotyping-by-sequencing.

BACKGROUND: One of the reasons hard red winter wheat cultivar 'Duster' (PI 644016) is widely grown in the southern Great Plains is that it confers a consistently high level of resistance to biotype GP of Hessian fly (Hf). However, little is known about the genetic mechanism underlying Hf resistance in Duster. This study aimed to unravel complex structures of the Hf region on chromosome 1AS in wheat by using genotyping-by-sequencing (GBS) markers and single nucleotide polymorphism (SNP) markers. RESULTS: Doubled haploid (DH) lines generated from a cross between two winter wheat cultivars, 'Duster' and 'Billings' , were used to identify genes in Duster responsible for effective and consistent resistance to Hf. Segregation in reaction of the 282 DH lines to Hf biotype GP fit a one-gene model. The DH population was genotyped using 2,358 markers developed using the GBS approach. A major QTL, explaining 88% of the total phenotypic variation, was mapped to a chromosome region that spanned 178 cM and contained 205 GBS markers plus 1 SSR marker and 1 gene marker, with 0.86 cM per marker in genetic distance. The analyses of GBS marker sequences and further mapping of SSR and gene markers enabled location of the QTL-containing linkage group on the short arm of chromosome 1A. Comparative mapping of the common markers for the gene for QHf.osu-1A (d) in Duster and the Hf-resistance gene for QHf.osu-1A (74) in cultivar '2174' showed that the two Hf resistance genes are located on the same chromosome arm 1AS, only 11.2 cM apart in genetic distance. The gene at QHf.osu-1A (d) in Duster has been delimited within a 2.7 cM region. CONCLUSION: Two distinct resistance genes exist on the short arm of chromosome 1A as found in the two hard red winter cultivars, 2174 and Duster. Whereas the Hf resistance gene in 2174 is likely allelic to one or more of the previously mapped resistance genes (H9, H10, H11, H16, or H17) in wheat, the gene in Duster is novel and confers a more consistent phenotype than 2174 in response to biotype GP infestation in controlled-environment assays.

Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes.

KEY MESSAGE: A stable QTL that may be used in marker-assisted selection in wheat breeding programs was detected for yield, yield components and drought tolerance-related traits in spring wheat association mapping panel. Genome-wide association mapping has become a widespread method of quantitative trait locus (QTL) identification for many crop plants including wheat (Triticum aestivum L.). Its benefit over traditional bi-parental mapping approaches depends on the extent of linkage disequilibrium in the mapping population. The objectives of this study were to determine linkage disequilibrium decay rate and population structure in a spring wheat association mapping panel (n = 285-294) and to identify markers associated with yield and yield components, morphological, phenological, and drought tolerance-related traits. The study was conducted under fully irrigated and rain-fed conditions at Greeley, CO, USA and Melkassa, Ethiopia in 2010 and 2011 (five total environments). Genotypic data were generated using diversity array technology markers. Linkage disequilibrium decay rate extended over a longer genetic distance for the D genome (6.8 cM) than for the A and B genomes (1.7 and 2.0 cM, respectively). Seven subpopulations were identified with population structure analysis. A stable QTL was detected for grain yield on chromosome 2DS both under irrigated and rain-fed conditions. A multi-trait region significant for yield and yield components was found on chromosome 5B. Grain yield QTL on chromosome 1BS co-localized with harvest index QTL. Vegetation indices shared QTL with harvest index on chromosome 1AL and 5A. After validation in relevant genetic backgrounds and environments, QTL detected in this study for yield, yield components and drought tolerance-related traits may be used in marker-assisted selection in wheat breeding programs.

The genetics of Ug99 stem rust resistance in spring wheat variety 'Linkert'.

Wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt) threatens wheat production worldwide. The objective of this study was to characterize wheat stem rust resistance in 'Linkert', a variety with adult plant resistance effective to emerging wheat stem rust pathogen strain Ug99. Two doubled haploid (DH) populations and one recombinant inbred line (RIL) population were developed with 'Linkert' as a stem rust resistant parent. Hard red spring wheat variety 'Forefront' and genetic stock 'LMPG' were used as stem rust susceptible parents of the DH populations. Breeding line 'MN07098-6' was used as a susceptible parent of the RIL population. Both DH and RIL populations with their parents were evaluated both at the seedling stage and in the field against Pgt races. Genotyping data of the DH populations were generated using the wheat iSelect 90k SNP assay. The RIL population was genotyped by genotyping-by-sequencing. We found QTL consistently associated with wheat stem rust resistance on chromosome 2BS for the Linkert/Forefront DH population and the Linkert/MN07098-6 RIL population both in Ethiopia and Kenya. Additional reliable QTL were detected on chromosomes 5BL (125.91 cM) and 4AL (Sr7a) for the Linkert/LMPG population in Ethiopia and Kenya. Different QTL identified in the populations reflect the importance of examining the genetics of resistance in populations derived from adapted germplasm (Forefront and MN07098-6) in addition to a genetic stock (LMPG). The associated markers in this study could be used to track and select for the identified QTL in wheat breeding programs.

Single amino acid change alters specificity of the multi-allelic wheat stem rust resistance locus SR9.

Most rust resistance genes thus far isolated from wheat have a very limited number of functional alleles. Here, we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) are characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation are used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1 and Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differs from SR9B by a single amino acid. SR9B and SR9G resistance proteins are also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences. Together, our results show that functional diversification of allelic variants at the SR9 locus involves single and multiple amino acid changes that recognize isolates of wheat stem rust.

Genome-wide association mapping for field and seedling resistance to the emerging Puccinia graminis f. sp. tritici race TTRTF in wheat.

Stem rust of wheat (Triticum spp.), caused by Puccinia graminis f. sp. tritici (Pgt), is one of the most impactful wheat diseases because of its threat to global wheat production. While disease mitigation has primarily been achieved through the deployment of resistant wheat varieties, emerging new virulent races continue to pose risks to the crop. For example, races such as Ug99 (TTKSK), TKTTF, and TTRTF have caused epidemics in different wheat growing regions of the world in recent years. A continual search for new and effective sources of resistance is therefore necessary to safeguard wheat production. This study assessed a breeding panel from the Ethiopian Institute of Agricultural Research (EIAR) wheat breeding program for seedling and field plant resistance to TTRTF and reports genomic regions conferring resistance to TTRTF. Trait correlations (r) were medium to strong (range = .38-.71) and heritabilities were moderate (.32-.56). Association analysis for resistance to TTRTF resulted in detection of 20 markers in 11 chromosomes; the marker S1B_175439851 was associated with resistance at both seedling and adult plant stages. Models with two to four QTL combinations reduced seedling and field disease severity by 12-48 and 9-17%, respectively. Genomic prediction for TTRTF resistance resulted in low to moderately-high predictions (mean correlations of .25-.47). Identification of resistant lines and QTL in the EIAR population is expected to assist in selection toward improved resistance to TTRTF. Specifically, the application of genomic selection (GS) in identifying resistant lines in future related breeding populations will further assist breeding efforts against this new stem rust pathogen race.

Field resistance to wheat stem rust in durum wheat accessions deposited at the USDA National Small Grains Collection.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re-emerging disease, posing a significant threat to durum wheat production worldwide. The limited number of stem rust resistance genes in modern cultivars compels us to identify and incorporate new effective genes in durum wheat breeding programs. We evaluated 8,245 spring durum wheat accessions deposited at the USDA National Small Grains Collection (NSGC) for resistance in field stem rust nurseries in Debre Zeit, Ethiopia and St. Paul, MN (USA). A higher level of disease development was observed at the Debre Zeit nursery compared with St. Paul, and the effective alleles of Sr13 in this nursery did not display the level of resistance observed at the St. Paul nursery. Four hundred and ninety-one (∽6%) accessions exhibited resistant to moderately susceptible responses after three field evaluations at Debre Zeit and two at St. Paul. Nearly 70% of these accessions originated from Ethiopia, Mexico, Egypt, and USA. Eight additional countries, namely Portugal, Turkey, Italy, Canada, Chile, Australia, Syria, and Tunisia contributed to 19% of the resistant to moderately susceptible entries. Among the 491 resistant to moderately susceptible accessions, 53.8% (n = 265) were landraces, and 28.4% (n = 139) and 11.4% (n = 55) were breeding lines and cultivars, respectively. Breeding lines and cultivars displayed a higher level and frequency of resistance than the landraces. We concluded that a large number of durum wheat accessions from diverse origins deposited at the NSGC can be exploited for diversifying and improving stem rust resistance in wheat.

Association mapping of resistance to emerging stem rust pathogen races in spring wheat using genotyping-by-sequencing.

The identification and characterization of resistance genes should outpace the rapid emergence of new P. graminis f. sp. tritici races, such as TTRTF and TTKTT, to mitigate stem rust damage to wheat. The objective of the current study was to identify and characterize P. graminis f. sp. tritici race resistance association signals. A total of 250 North American spring wheat lines were evaluated at the seedling stage with a total of seven isolates including TKKTP, TKTTF, TKTTF, TRTTF, TTRTF, TTKSK, and TTKTT. The lines were genotyped by a GBS platform and 9,042 SNPs were used for identification of chromosome regions associated with resistance against the seven isolates. Strong association signals were detected on chromosomes 6BL (Sr11 gene region) and 4AL, likely Sr7a, for resistance against both TKKTP and TKTTF. Similarly, association signals were also detected on chromosomes 4AL (race TTRTF resistance) and 4BS (race TTKSK and TTKTT resistance). Association analysis based on mean phenotypic differences between closely related isolates identified QTL that were not elucidated by direct association mapping of the responses, individually. Overall, with the exception of race TRTTF, each race shared at least one association signal with another race. However, the number of race-specific association signals are larger than that of association signals common among races suggesting the need for identifying and characterizing QTL/genes for newly emerging stem rust pathogen races. There was also high concordance between PCA-based GWAS association signals and association signals from that of both single and multi-locus mixed models.

Mapping and Validation of Stem Rust Resistance Loci in Spring Wheat Line CI 14275.

Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) remains a constraint to wheat production in East Africa. In this study, we characterized the genetics of stem rust resistance, identified QTLs, and described markers associated with stem rust resistance in the spring wheat line CI 14275. The 113 recombinant inbred lines, together with their parents, were evaluated at the seedling stage against Pgt races TTKSK, TRTTF, TPMKC, TTTTF, and RTQQC. Screening for resistance to Pgt races in the field was undertaken in Kenya, Ethiopia, and the United States in 2016, 2017, and 2018. One gene conferred seedling resistance to race TTTTF, likely Sr7a. Three QTL were identified that conferred field resistance. QTL QSr.cdl-2BS.2, that conferred resistance in Kenya and Ethiopia, was validated, and the marker Excalibur_c7963_1722 was shown to have potential to select for this QTL in marker-assisted selection. The QTL QSr.cdl-3B.2 is likely Sr12, and QSr.cdl-6A appears to be a new QTL. This is the first study to both detect and validate an adult plant stem rust resistance QTL on chromosome arm 2BS. The combination of field QTL QSr.cdl-2BS.2, QSr.cdl-3B.2, and QSr.cdl-6A has the potential to be used in wheat breeding to improve stem rust resistance of wheat varieties.

Bulked segregant analysis RNA-seq (BSR-Seq) validated a stem resistance locus in Aegilops umbellulata, a wild relative of wheat.

Many disease resistance genes that have been transferred from wild relatives to cultivated wheat have played a significant role in wheat production worldwide. Ae. umbellulata is one of the species within the genus Aegilops that have been successfully used as sources of resistance genes to leaf rust, stem rust and powdery mildew. The objectives of the current work was to validate the map position of a major QTL that confers resistance to the stem rust pathogen races Ug99 (TTKSK) and TTTTF with an independent bi-parental mapping population and to refine the QTL region with a bulk segregant analysis approach. Two F2 bi-parental mapping populations were developed from stem rust resistant Ae. umbellulata accessions (PI 298905 and PI 5422375) and stem rust susceptible accessions (PI 542369 and PI 554395). Firstly, one of the two populations was used to map the chromosome location of the resistance gene. Later on, the 2nd population was used to validate the chromosome location in combination with a bulk segregant analysis approach. For the bulk segregant analysis, RNA was extracted from a bulk of leaf tissues of 12 homozygous resistant F3 families, and a separate bulk of 11 susceptible homozygous F3 families derived from the PI 5422375 and PI 554395 cross. The RNA samples of the two bulks and the two parents were sequenced for SNPs identification. Stem rust resistance QTL was validated on chromosome 2U of Ae. umbellulata in the same region in both populations. With bulk segregant analysis, the QTL position was delimited within 3.2 Mbp. Although there were a large number of genes in the orthologous region of the detected QTL on chromosome 2D of Ae. tauschii, we detected only two Ae. umbellulata NLR genes which can be considered as a potential candidate genes.

A Genome-Wide Association Study of Field and Seedling Response to Individual Stem Rust Pathogen Races Reveals Combinations of Race-Specific Genes in North American Spring Wheat.

Stem rust of wheat caused by the fungal pathogen Puccinia graminis f. sp. tritici historically caused major yield losses of wheat worldwide. To understand the genetic basis of stem rust resistance in contemporary North American spring wheat, genome-wide association analysis (GWAS) was conducted on an association mapping panel comprised of 250 elite lines. The lines were evaluated in separate nurseries each inoculated with a different P. graminis f. sp. tritici race for 3 years (2013, 2015, and 2016) at Rosemount, Minnesota allowing the evaluation of race-specificity separate from the effect of environment. The lines were also challenged with the same four races at the seedling stage in a greenhouse facility at the USDA-ARS Cereal Disease Laboratory. A total of 22,310 high-quality SNPs obtained from the Infinium 90,000 SNPs chip were used to perform association analysis. We observed often negative and sometimes weak correlations between responses to different races that highlighted the abundance of race-specific resistance and the inability to predict the response of the lines across races. Markers strongly associated with resistance to the four races at seedling and field environments were identified. At the seedling stage, the most significant marker-trait associations were detected in the regions of known major genes (Sr6, Sr7a, and Sr9b) except for race QFCSC where a strong association was detected on chromosome arm 1AL. We postulated the presence of Sr2, Sr6, Sr7a, Sr8a, Sr9b, Sr11, Sr12, Sr24, Sr25, Sr31, and Sr57 (Lr34) in this germplasm based on phenotypic and marker data. We found over half of the panel possessed three or more Sr genes, and most commonly included various combinations of Sr6, Sr7a, Sr8a, Sr9b, Sr11, Sr12, and Sr57. Most of these genes confer resistance to specific P. graminis f. sp. tritici races accounting for the prevalent stem rust resistance in North American spring wheat.

Genotyping-by-Sequencing Facilitates a High-Density Consensus Linkage Map for Aegilops umbellulata, a Wild Relative of Cultivated Wheat.

High-density genetic maps are useful to precisely localize QTL or genes that might be used to improve traits of nutritional and/or economical importance in crops. However, high-density genetic maps are lacking for most wild relatives of crop species, including wheat. Aegilops umbellulata is a wild relative of wheat known for its potential as a source of biotic and abiotic stress resistance genes. In this work, we have developed a framework consensus genetic map using two biparental populations derived from accessions PI 298905, PI 542369, PI 5422375, and PI 554395. The framework map comprised 3009 genotype-by-sequence SNPs with a total map size of 948.72 cM. On average, there were three SNPs per centimorgan for each chromosome. Chromosome 1U was the shortest (66.5 cM), with only 81 SNPs, whereas the remaining chromosomes had between 391 and 591 SNP markers. A total of 2395 unmapped SNPs were added to the linkage maps through a recombination frequency approach, and increased the number of SNPs placed on the consensus map to a total of 5404 markers. Segregation distortion was disproportionally high for chromosome 1U for both populations used to construct component linkage maps, and thus segregation distortion could be one of the probable reasons for the exceptionally reduced linkage size for chromosome 1U. From comparative analysis, Aeumbellulata chromosomes except 4U showed moderate to strong collinearity with corresponding homeologous chromosomes of hexaploid wheat and barley. The present consensus map may serve as a reference map in QTL mapping and validation projects, and also in genome assembly to develop a reference genome sequence for Ae. umbellulata.

Application of Population Sequencing (POPSEQ) for Ordering and Imputing Genotyping-by-Sequencing Markers in Hexaploid Wheat.

The advancement of next-generation sequencing technologies in conjunction with new bioinformatics tools enabled fine-tuning of sequence-based, high-resolution mapping strategies for complex genomes. Although genotyping-by-sequencing (GBS) provides a large number of markers, its application for association mapping and genomics-assisted breeding is limited by a large proportion of missing data per marker. For species with a reference genomic sequence, markers can be ordered on the physical map. However, in the absence of reference marker order, the use and imputation of GBS markers is challenging. Here, we demonstrate how the population sequencing (POPSEQ) approach can be used to provide marker context for GBS in wheat. The utility of a POPSEQ-based genetic map as a reference map to create genetically ordered markers on a chromosome for hexaploid wheat was validated by constructing an independent de novo linkage map of GBS markers from a Synthetic W7984 × Opata M85 recombinant inbred line (SynOpRIL) population. The results indicated that there is strong agreement between the independent de novo linkage map and the POPSEQ mapping approach in mapping and ordering GBS markers for hexaploid wheat. After ordering, a large number of GBS markers were imputed, thus providing a high-quality reference map that can be used for QTL mapping for different traits. The POPSEQ-based reference map and whole-genome sequence assemblies are valuable resources that can be used to order GBS markers and enable the application of highly accurate imputation methods to leverage the application GBS markers in wheat.